位于质体小球的ABC1K激酶调控柑橘果肉中类胡萝卜素积累的机理研究

The plastoglobule within chromoplasts is the main site for carotenoids biosynthesis and accumulation. The plastoglobule-localized ABC1Ks (Activity of bc1 complex kinases) are one of the most abundant proteins in the plastoglobule, and have been suggested to play an important role in plastoglobule development and accumulation of specific secondary metabolites including carotenoids in chromoplasts. Our previous studies showed that plastoglobule-localized Citrus ABC1K3 increase significantly at both the mRNA and the protein level during sweet orange ripening. Furthermore, overexpression of CitABC1K3 in citrus callus or tomato (Ailsa Craig) results in the formation of yellowish transgenic tissues indicative of accumulation of specific carotenoids. However, how ABC1Ks exert their regulatory functions is poorly understood. The overall goal of this project is to understand the role of CitABC1K3 in regulating plastoglobules development and carotenoid accumulation in citrus fruits. There are four specific aims: (i) Determine the biological functions of CitABC1K3 via RNAi and overexpression of the relevant DNA constructs in citrus callus, tomato, and Hongkong kumquat (Fortunella hindsii Swingle; a citrus species with a short juvenile period); (ii) the biochemical function of CitABC1K3 in carotenoids accumulation will be conducted by enzymatic assays by using transfection the CitABC1K3 to E. coli strains; (iii) Identify CitABC1K3-interacting proteins using co-immunoprecipitation, yeast two-hybrid screens, and bimolecular fluorescence complementation; (iv) Identify protein substrates of CitABC1K3 by phosphorylation assays of candidate CitABC1K3-interacting proteins. The expected results from this work will help clarify the functional roles of CitABC1K3 in plastoglobule development and color formation in sweet oranges, therefore providing insight into the molecular basis of citrus fruit coloration that is valuable for genetic improvement in future breeding programs.

质体小球是果肉细胞有色体中合成与积累类胡萝卜素的主要器官，其数量和大小对果实的色泽品质至关重要。前期研究结果初步表明，位于质体小球的ABC1Ks激酶家族中的CitABC1K3可能通过影响质体小球的发育而参与类胡萝卜素的代谢调控，但其互作及调控机制尚不清楚。本项目在前期研究的基础上，拟先在柑橘愈伤，番茄和短童期的山金柑中进行超量和沉默表达，评价其对质体小球的发育和果实色泽形成的影响，以验证CitABC1K3基因的功能；其次，采用类胡萝卜素工程菌体系明确CitABC1K3的作用底物及其产物；同时，采用免疫共沉淀结合质白质谱鉴定的方法寻找与CitABC1K3互作的蛋白，并利用酵母双杂交实验、双分子荧光互补实验明确它们的互作关系，构建其互作网络；最后，采用体内和体外的蛋白磷酸化修饰手段探索其修饰底物，进一步明确CitABC1K3调控质体小球发育及类胡萝卜素形成的机理，为柑橘果色改良和调控奠定基础。

有色体中的质体小球是合成与积累类胡萝卜素的主要器官，其数量和大小对果实的色泽品质至关重要。本项目针对柑橘果肉有色体中质体小球定位的蛋白ABC1K3和PES进行功能验证，主要取得了以下研究进展：1）明确了ABC1K3参与质体小球的形成和色素的积累。亚细胞定位实验表明ABC1K3与质体小体标记蛋白PGL35共定位于质体小球中；将ABC1K3稳定转化到番茄中，可使红番茄变微黄。在番茄转基因超量表达系中，b-胡萝卜素和叶黄素的组成比例比对照系更高，这些色素的积累是导致果实变黄的原因，并在柑橘愈伤中也得到类似的验证。BIFC等互作实验表明ABC1K3与FBN1存在蛋白之间的互作关系。关于ABC1K3基因的功能及其与FBN1共同参与质体小球的形成和色素的积累正在深入研究。2）建立了一种提纯柑橘有色体中质体小球的方法（专利申请号2016108456990）。同时，由于柑橘果皮的造油体中含有大量的质体小球，我们利用梯度离心分离出造油体。对以上分离的质体小球和造油体，通过液相-质谱串联技术，我们分别鉴定到的39个质体小球候选蛋白（Horticulture Research, 2018; 5:6）。3）基于质体小球的脂质组代谢组和蛋白质组学，以及转基因功能验证，研究表明质体小球定位的PES蛋白参与了类胡萝卜素酯化的功能。4）发现了三种可促进果实表面着色的方法，并申请了国家发明专利（专利申请号2017101929485.0、201710450823.8、201710453537.7）。以上研究结果为柑橘果实类胡萝卜素的稳定，品种改良奠定基础。